The primary objective of this project is to understand the signaling mechanism of light activated sensory rhodopsins (SRs), part of the growing family of 7-helix transmembrane microbial rhodopsins. Examples of SRs include SRI and SRII from archaebacteria, which control phototaxis, Anabaena sensory rhodopsin (ASR) from freshwater cyanobacteria, which functions as photochromic sensors, some forms of proteorhodopsin (PR) found in marine bacteria, which control a variety of cellular functions, and the recently discovered channel-rhodopsins (ChRs), which control phototactic and photophobic responses in green algae. In contrast to bacteriorhodopsin (BR), the well-studied light-driven proton pump, most SRs function by transmitting a signal to an associated transducer protein, analogous to the well-known G-proteins in the rhodopsin signaling cascade. Still others, such as ChRs, convey a signal by opening a self-contained light-activated ion channel. Detailed knowledge at the molecular level of the signaling mechanisms of SRs would be of great significance for understanding a variety of membrane protein-based cellular processes as well as have applications in the field of biotechnology and biomedicine. In the case of SRII from Natronobacterium pharaonis, the high-resolution structure of the receptor linked to the transmembrane part of its cognate HtrII transducer has revealed important molecular details of the protein- protein interactions, including the contact residues and internal water molecules located in the interface region. However, so far X-ray diffraction has not revealed the molecular events connecting the initial light-induced isomerization of the retinal chromophore to the activation of the transducer, possibly due to structural constraints imposed by the crystal lattice. In the case of other SRs, even less information is known due to difficulties of crystallization and expression. In addition, our own and other studies demonstrate the importance of studying SRs under physiological conditions in native membranes. Ideally, new techniques are needed for studying SR structural changes in a native environment, including even the inside the cell. In this project we will use an array of advanced IR-based techniques, some of which have recently been developed in our laboratory, to examine the detailed molecular events which lead to signal activation in SRs. Significant progress has been made in the past grant period leading to new molecular details and tentative models of SR function. In the proposed research, these models will be tested in detail by measuring structural changes of specific residues, internal water molecules, and the peptide backbone in SR receptor-transducer complexes on a time-scale of sub-picoseconds to seconds. A unique aspect of the proposed studies is the ability to, for the first time, study these structural changes in intact functioning cells where direct correlation with other events, such as phototaxis and photoinduced charge movements, can be measured. The proposed studies will also benefit from our development of new methods to i) measure sub-picosecond structural changes in the protein and its internal water molecules using advanced ultrafast time-resolved IR spectroscopy, ii) rapidly express and isotope label SRs and their transducer complexes using the technology of cell-free expressed nanolipoparticles (NLPs), and iii) measure time-resolved FTIR-differences of SRs in single crystals. This work will be facilitated by close collaborations with the laboratories of Dr. J. Spudich at the University of Texas Medical Center, Houston, whose laboratory has contributed much of our current knowledge about SRs, and Dr. M. Coleman at the Lawrence Livermore National Laboratories, whose group has developed cell-free techniques to express membrane proteins in NLPs. Specifi objectives of this project are: PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the signaling mechanism of light activated sensory rhodopsins (SRs). Most SRs function by transmitting a signal to an associated transducer protein. In contrast, channel-rhodopsins convey a signal by opening a self-contained light-activated ion channel. SRs provide an important opportunity to understand how evolution has modified similar membrane protein structures to accomplish very different molecular mechanisms of signaling. In this project we will use an array of advanced IR-based techniques to examine the detailed molecular events which lead to signal activation in SRs including new methods to study these proteins inside living cells.